Wnts are secreted growth factors that specify cell fate during embryogenesis and renewal of tissues in the adult. Inappropriate activation of the pathway is associated with a number of cancers. Wnts bind to two co- receptors: 7-transmembrane helix receptors called Frizzled proteins (Frz), and single pass transmembrane receptors called LDL-receptor related proteins 5 and 6 (Lrp5/6). Activation of Lrp5 or Lrp6 leads to phosphorylation of its intracellular domain and transmission of the Wnt signal. Wnt-Frz-Lrp5/6 interactions are modulated by various activators and inhibitors, including the vertebrate Dickkopf (Dkk) proteins. In this proposal, biochemical, structural, and biophysical analyses are used to define the mechanism and specificity of Lrp5/6 interactions with Wnt pathway activators and inhibitors. The results will provide a mechanistic underpinning for future efforts to modulate Wnt signaling therapeutically. 1. To define how Dkk1 modulates the conformation of Lrp6 to render it inactive in Wnt signaling, crystal structures of human Dkk1 bound different portions of Lrp6 will be determined, and the energetics of these interactions will be determined accurately by calorimetry. 2. The activated state of Lrp6 will be investigated by determining structures of Lrp6 bound to activators, including a recently described monoclonal antibody, and by determining energetic contributions of different regions of Lrp6 to these interactions. 3. Mutations of Lrp6 residues identified as important Dkk1 interaction residues by structure-based mutagenesis will be tested for binding to purified Wnt3a, in order to map out the Wnt3a binding surface on Lrp6. Structural studies will be performed on Drosophila WntD to define surfaces in the homologous classical Wnts important for receptor binding.